The present invention relates to a method for producing particles, and more particularly related to a method for producing a cobalt-protein complex containing a cobalt particle and its related technologies.
In recent years, there have been vigorous studies in the field of bioelectronics which is a combination of biotechnology and electronics. As a result of such studies, biosensors using proteins such as enzymes, or like devices have been already practically used.
As an attempt of the application of biotechnology to other fields, there is a study of incorporating particles composed of a metal or a metal compound into apoferritin which is a protein having the function of holding a metal compound, thereby producing particles having a uniform diameter at the nano-order level. In order to introduce various kinds of metals, metal compounds or the like into apoferritin according to various applications, many researches have been carried out.
Hereinafter, description of apoferritin will be given. Apoferritin is a protein which is extensively present in the biological world. It has the function of adjusting the amount of iron which is a necessary micronutrient element in a living body. A complex of iron or an iron compound with apoferritin is called “ferritin”. When the amount of iron exceeds a necessary level in a living body, iron could be harmful. So, excessive iron is stored in a living body in the form of ferritin. Ferritin releases iron ions, when necessary, and then it becomes apoferritin again.
FIG. 1 is a schematic view of the structure of apoferritin. As shown in FIG. 1, apoferritin 1 is a globular protein in which 24 monomer subunits each being composed of a polypeptide chain assemble and form non-covalent bonds and which has a molecular weight of about 460,000. The globular protein has a diameter of about 12 nm and exhibits higher thermal stability and higher pH stability than normal proteins. The apoferritin 1 has a hollow-like holding portion 4 having a diameter of about 6 nm in the center. The holding portion 4 is connected to the outside via a channel 3. For example, when ferric iron ions are incorporated into the apoferritin 1, the iron ions enter the apoferritin 1 through the channel 3 and are oxidized in a portion called the ferrooxidase center in one of the subunits. Then, they reach the holding portion 4 and finally are condensed in a negative charge region located in the inside surface of the holding portion 4. Then, 3000-4000 iron atoms assemble and are held in the holding portion 4 in the crystalline form of ferrihydride (5Fe2O3.9H2O). A particle containing metal atoms held in the holding portion 4 has the almost same diameter as that of the holding portion 4, i.e., about 6 nm.
Using apoferritin, a complex of apoferritin with particles artificially made to contain a metal other than iron or a metal compound is produced.
Up until today, there have been reports of introductions of metals or metal compounds into apoferritin, such as introduction of manganese into apoferritin (P. Mackle, 1993, J. Amer. Chem. Soc. 115, 8471-8472; F. C. Meldrum et al., 1995, J. Inorg. Biochem. 58, 59-68), introduction of uranium into apoferritin (J. F. Hainfeld, 1992, Proc. Natil. Acad. Sci. USA 89, 11064-11068), introduction of beryllium into apoferritin (D. J. Price, 1983, J. Biol. Chem. 258, 10873-10880), introduction of aluminum into apoferritin (J. Fleming, 1987, Proc. Natl. Acad. Sci. USA, 84, 7866-7870), and introduction of zinc into apoferritin (D. Price and J. G. Joshi, Proc. Natl. Acd. Sci. USA, 1982, 79, 3116-3119). Particles composed of any one of these metals or metal compounds have also about the same diameter as that of the holding portion 4 of apoferritin, i.e., about 6 nm.
Processes by which a particle containing iron atoms is formed in the natural world will be briefly described hereinafter.
On the surface of the channel 3 (see FIG. 1) connecting the outside and inside of the apoferritin 1, amino acid residues with a negative charge at pH 7-8 are exposed and Fe2+ ions with a positive charge are incorporated into the channel 3 through electrostatic interaction.
Also, on the inside surface of the holding portion 4 of the apoferritin 1, many glutamic acid residues which are amino acid residues and have a negative charge at pH 7-8 are exposed as on the inside surface of the channel 3. Fe2+ ions incorporated through the channel 3 are oxidized at the ferroxidase center and then are introduced to the holding portion 4 located at the inside of the apoferritin 1. Iron ions are condensed through electrostatic interaction, and then nucleation of ferrihydride (5Fe2O3.9H2O) crystals occurs.
Thereafter, increasingly incorporated iron ions are adhered to the nucleus of a ferrihydride crystal and the nucleus composed of iron oxide is grown. Thus, particles with a diameter of 6 nm are formed in the holding portion 4. This is how iron ions are incorporated and particles composed of iron oxide is formed.
The mechanism of incorporation of iron ions into apoferritin has been described. However, since ions of any other metals which have been reported regarding introduction thereof into apoferritin have a positive charge, ions are incorporated into apoferritin by almost the same mechanism as that for iron ions.